Cell specific nuclear antigens of boar spermatozoa.
Demembranated boar sperm heads were differentially extracted at conditions involving high salt-urea, proteolysis and DNase I cleavage that mimic the conditions promoting the in vivo decondensation of the fertilizing sperm nucleus in the egg ooplasm. The sperm-unique subset of proteins was studied which remained bound in the residual salt-resistant nuclear structure operationally defined as sperm nuclear matrix. By means of polyvalent antisera the immune specificity of the sperm nucleoprotein complex was estimated using ELISA and microcomplement fixation test as compared to somatic type dehistonized chromatin of boar liver. To define immunologically specific sperm DNA-associated proteins hybridomas were generated by fusing lymphocytes immunized with boar sperm protein/DNA complex. Monoclonal antibodies were selected (Mab 1A8, 1B3, 2B5, 2H5 and 3A4) which identified protein moieties in the sperm DNA-tight binding proteins complex resistant to cleavage with DNase I and sensitive upon digestion with high concentration of proteases. No appreciable reactivity was recorded of the antibodies to somatic chromatin and no significant binding to ssDNA. A polypeptide in the residual sperm nuclear structure of apparent Mr 27 kDa was recognized by Mab 3A4 as detected by Western blotting. The enhanced reactivity to the DNase I digested sperm nuclear fraction (except for Mab 2H5) suggests that DNA protected from nuclease digestion by a protein might be essential for immune reactivity and full antigenic integrity as well as the dependence of the cognate proteins on the binding to DNA for antigenicity and immune specificity. The functioning of the identified putative sperm specific proteins is anticipated in the structural rearrangement of chromatin in the zygote.
Batova
I N
IN
Department of Immunobiology of Reproduction, Bulgarian Academy of Sciences, Sofia, Bulgaria.
Kyurkchiev
S D
SD
Kehayov
I R
IR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D034961:Antigens, Nuclear; D015153:Blotting, Western / Q000662:veterinary; D002843:Chromatin / Q000276:immunology; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis / Q000276:immunology*; D003168:Complement Fixation Tests / Q000662:veterinary; D003850:Deoxyribonuclease I / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D000939:Epitopes / Q000032:analysis / Q000276:immunology; D005260:Female; D006822:Hybrid Cells; D007106:Immune Sera / Q000276:immunology; D008099:Liver / Q000276:immunology; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence / Q000662:veterinary; D015530:Nuclear Matrix / Q000737:chemistry; D009687:Nuclear Proteins / Q000032:analysis / Q000276:immunology*; D011817:Rabbits; D013077:Sperm Head / Q000276:immunology* / Q000502:physiology / Q000648:ultrastructure; D013552:Swine / Q000502:physiology
D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D000939:Epitopes; D007106:Immune Sera; D009687:Nuclear Proteins; D003850:Deoxyribonuclease I